This invention relates to corn (Zea mays L.) seed and grain having a significantly higher oleic acid content by virtue of heritable genes for increased oil and oleic acid content and/or lowered levels of linoleic acid. The present invention also relates to the production of high oil, high oleic grain, plants and plant parts grown from such grain and uses of such improved grain.
Corn oil is comprised primarily of even-numbered carbon chain fatty acids. The distribution of fatty acids in typical corn oil is approximately 12% palmitic acid (16:0), 2% stearic acid (18:0), 25% oleic acid (18:1), 60% linoleic acid (18:2), and 1% linolenic acid (18:3). Palmitic and stearic acids are referred to as saturated fatty acids because their carbon chains contains only single bonds and the carbon chain is xe2x80x9csaturatedxe2x80x9d with hydrogen atoms. Oleic, linoleic, and linolenic acids contain one, two, and three double bonds respectively, and are referred to as unsaturated fatty acids. Fatty acids in corn oil nearly always occur esterified to the hydroxyl groups of glycerol, thus forming triglycerides. Approximately 99% of refined corn oil is made up of triglycerides; Corn Oil, Corn Refiners Association, Inc., 1001 Connecticut Ave., N.W., Washington, D.C. 20036, 1986, 24 pp.
When exposed to air, unsaturated fatty acids are subject to oxidation which causes the oil to have a rancid odor. Oxidation is accelerated by high temperatures, such as in frying conditions. The rate of oxidation is proportional to the number of double bonds in the fatty acids. Thus, linoleic acid with two double bonds is more unstable than oleic acid which has only one double bond. Oxidation reduces the shelf life of products containing corn oil because of the oil""s high proportion of linoleic acid. Corn oil and products containing corn oil are often packaged under nitrogen in special packaging materials such as plastic or laminated foil, or are stored under refrigeration to extend their shelf life. These extra measures to reduce oxidation and subsequent rancidity add considerable cost to products containing corn oil.
Another measure to reduce the effects of oxidation on corn oil is to chemically hydrogenate the oil. This commercially important process by which hydrogen is added to double bonds of unsaturated fatty acids changes the physical properties of the oil and extends the shelf life of products containing corn oil. Hydrogenated vegetable oils are used to make margarine, salad dressings, cooking oils, and shortenings, for example. Approximately half a billion pounds, or roughly 40-50% of corn oil produced in the U.S. is used for cooking and for salad oils; Fitch, B., JAOCS, 1985, Vol. 62, no. 11, pp. 1524-31. Production of a more stable oil by genetic means would clearly have value by reducing or eliminating the time and input costs of chemical hydrogenation.
In addition to the economic factors associated with chemical hydrogenation of corn oil, there are human health factors that favor the production of a natural high oleic oil. During the hydrogenation process, double bonds in fatty acids are completely hydrogenated or are converted from the cis configuration to the trans configuration. Cis double bonds cause a fatty acid molecule to bend, which impairs crystallization and keeps the oil liquid at room temperature. During hydrogenation, cis bonds are straightened into the trans configuration, causing the oil to harden at room temperature. Recent studies on the effect of dietary trans fatty acids on cholesterol levels show that the trans isomer of oleic acid raises blood cholesterol level at least as much as saturated fatty acids, which have been know for some time to raise cholesterol in humans; Mensink, R. P. and B. K. Katan, N. Engl. J. Med., 1990, 323:439-45. Furthermore, the studies show that the undesirable low density lipoprotein level increases and the desirable high density lipoprotein level decreases in response to diets high in trans fatty acids. Large amounts of trans fatty acids are found in margarines, shortenings, and oils used for frying; the most abundant trans fatty acid in the human diet is the trans isomer of oleic acid, elaidic acid. A natural high oleic corn oil, which does not contain elaidic acid, will benefit consumers in general, and will particularly benefit those people who control their cholesterol level through their diet.
The human diet could also be improved by reducing saturated fat intake. Much of the saturated fat in the human diet comes from meat products. Poultry and swine diets often contain animal fat, which is high in saturated fatty acids, as an energy source. Non ruminant animals such as these are very susceptible to tissue fatty acid alteration through dietary modification; M. F. Miller, et al., J. Anim. Sci., 1990, 68:1624-31. A large portion of animal feed rations is made up of corn, which typically contains only about 4% oil. By replacing some or all of the supplemental animal fat in a feed ration with the oil present in high oil corn varieties, which contain up to 10% oil, it will be possible to produce meat products having less saturated fats. Feeding trials in which swine were fed diets high in oleic acid show that the amount of oleic acid deposited in adipose tissue can be raised substantially without adversely influencing the quality of the meat; M. F. Miller, et al., supra; L. C. St. John, et al., J. Anim. Sci., 1987, 64:1441-47. The degree of saturation of the fatty acids comprising an oil determines whether it is liquid or solid. In these studies, the animal diets high in oleic acid led to meat quality that was acceptable to the meat processing industry because of the low level of polyunsaturated fatty acids. Therefore, it can be extended that a feed ration containing high oleic, high oil corn would be preferable to one containing high oil corn which contains a high level of linoleic acid. Consumption of monounsaturated fatty acids decreases the LDL level without affecting the HDL level; Mattson, F. R., and S. M. Grundy, J. Lipid Res., 1985, 26:194. The HDL portion is responsible for removal of cholesterol from the body; L. C. St. John, supra. Processed meats produced from animals fed diets containing high oil, high oleic corn will be more healthful in the human diet.
The corn kernel is a product of double fertilization; Kiesselbach, T. A., 1980, The Structure and Reproduction of Corn, University of Nebraska Press. This means that both the diploid embryo (giving rise to the germ and seedling) and the triploid endosperm (the nutritive structure surrounding the germ) contain genes transmitted from both the male and female parents. Nonetheless, the genes affecting grain composition and quality are similar enough in most field corn inbreds that crossing any given female with a large variety of male plants does not result in dramatic changes in the compositional or quality characteristics of the resulting seed or grain. Likewise, planting different field corn hybrids within pollinating proximity to each other will not, in most cases, substantially affect the quality of the grain harvested on each type.
In contrast, a minority of commercial corn inbreds or hybrids do contain genes which substantially modify grain quality. These hybrids, include those containing the waxy gene. Such waxy gene hybrids must be isolated from normal, non-waxy corn inbreds or hybrids in order to recover waxy seed or grain. If a non-waxy pollen grain (as found in most field corn inbreds and hybrids) pollinates an ovule borne on a waxy inbred or hybrid, the resulting kernel will be non-waxy, even though adjacent kernels on the same ear, pollinated by waxy pollen, will remain waxy. This immediate effect of pollen genotype on kernel characteristics is termed xe2x80x9cxeniaxe2x80x9d, and the hybrid nature of such kernels is recognizable by particular phenotypic characteristics (color, shape, size, etc.) owing to the direct influence exerted by the genotype of the pollen; Rieger, R., A. Michaelis and M. M. Green, 1968, A Glossary of Genetics and Cytogenetics, Springer-Verlag, N.Y. This immediate effect of pollen genotype on grain quality has been observed with pollen obtained from high-oil corn plants; Alexander, D. E. and R. J. Lambert, 1968, Relationship of Kernel Oil Content to Yield in Maize Crop Science 8:272-274.
Production of oleic acid in corn is under genetic control, although the mode of,inheritance is only partially understood. Oil production in the kernel occurs primarily in the germ. Fatty acid biosynthesis is regulated by a multi-step biochemical pathway whereby the saturated fatty acids, palmitic and stearic, are synthesized and subsequently dehydrogenated to oleic, linoleic, and linolenic acids; Lipid Metabolism, In: Introduction to Plant Biochemistry, 2nd Ed., 1983, Pergamon Press, Goodwin and Mercer, Eds., pp 273-327. A single gene locus, designated ln, was reported to be responsible for regulating the levels of oleic and linoleic acids in corn; Poneleit, C. G., and D. E. Alexander, Science, 1965, 147:1585-86. Subsequent studies show that the mode of inheritance of oleic acid is more complicated than first thought. At least two loci have been shown to regulate the oleic acid level; de la Roche et al., Crop Sci., 1971, 11:856-59. In a study involving eight different reciprocal crosses and their parental inbred lines, it was concluded that inheritance of increased oleic content in corn can result from dominant, partially dominant, and even recessive gene action; Jellum, M. D., J. Hered., 1966, 57:243-44. Only one report has been found in which the inheritance of oleic acid in a high oil corn line, IHO, is discussed; de la Roche, et al., supra. The report states that the quality of corn oil increases as the linoleic acid content increases. The data are presented in terms of the linoleic acid content, which for IHO is reported to be approximately 47% of the oil fraction. From our studies of thousands of samples, there is an inverse relationship between oleic acid and linoleic acid content. A line that is 47% linoleic acid would contain 35-40% oleic acid, which is substantially less than the oleic content in the present invention. Also, IHO is not an agronomically acceptable line and would not be used in commercial production; Glover, D. V., and E. T. Mertz, Corn, In Nutritional Quality of Cereal Grains: Genetic and Agronomic Improvement, Agronomy Monograph no. 28, Copyright 1987, ASA-CSSA-SSSA, 677 South Segoe Road, Madison, Wis. 53711, USA, Chapter 7, pp. 183-336; Fitch, B., JAOCS, 1985, Vol. 62, no. 11, pp. 1524-31.
A survey of plant introductions for fatty acid profile shows that greater genetic diversity exists in corn of foreign origin than exists in U.S. corn; Jellum, M. D., 1970, J. Agr. Food Chem., 18:3, pp. 365-70. Oleic acid content ranged from 14 to 64% in the plant introductions screened, which represented germplasm from over 50 foreign countries. Plant introductions are a valuable source of genetic diversity for many traits, including oleic acid content. However, breeding genes from plant introductions of foreign origin into elite U.S. adapted inbred lines is a costly process requiring three to six years.
A breeding strategy know as recurrent selection has been suggested as a means of increasing the oleic acid level in corn; Poneleit, C. G., and L. F. Bauman, Crop Sci., 1970, 10:338-41. This breeding method was applied to maize plant introductions and is the basis for a patent application for high oleic corn products and methods for their production; PCT/US91/04626. To have commercial utility, the value of a trait, such as high oleic oil, must be worth more than the costs associated with production, storage, and shipment of the grain. A bushel of shelled corn, which weighs approximately 56 pounds, can yield approximately two pounds of oil when milled. Because of the small amount of oil normally found in corn, the added value of an improved oil, such as high oleic oil, is unlikely to be sufficient to pay for the production and identity preservation costs, unless substantially greater oil is produced as in newly developed high oil corn varieties.
To have utility in an animal feed ration as a means of improving carcass quality and subsequently improving the human diet, high oleic corn must be capable of supplying enough oleic acid in the diet to raise the oleic acid level in the meat. Corn is included in animal feed as the main source of energy, the majority of which comes from the high starch content of corn, and other sources of energy such as animal fat, vegetable fat, or animal-vegetable fat blends are commonly added to increase the energy density of feed rations. For example, the amount of corn oil included in the corn fraction of a typical commercial poultry feed ration is about 2.5% in a ration that contains 65-70% corn. To increase the energy density of feed rations, highly saturated animal fat or animal-vegetable fat blends are added at approximately 5 to 8% of the diet. High oil corn with an energy content which is significantly higher than that of normal corn can reduce or totally eliminate the use of or need for added fat when used in a typical poultry ration.
A typical chicken broiler corn-soybean meal diet supplemented with an animal-vegetable fat blend contains approximately 1.937% oleic acid. Increasing the oleic acid content of the oil contained in corn used in a feed ration from the 25% found in normal corn to 60% (also in a normal or low oil variety) increases the oleic acid in the feed ration to 2.733%. Increasing the oleic acid content from 25% to 60% of the oil present in high oil corn grain that contains 8-10% oil increases the oleic acid content of the feed ration to 4.266%. These increases represent a 30% increase in oleic acid content when normal corn is used in the feed ration, and a 120% increase when high oil corn varieties are used. High oil corn can reduce or totally eliminate the need for added fat when used in a typical poultry ration, suggesting that modifications to the fatty acid profile of corn oil need to be made in a high oil corn variety to have utility in improving carcass quality. The high oleic corn lines described in the aforementioned patent application are not high oil corn lines.
Most cereal crops are handled as commodities, and many of the industrial and animal feed requirements for these crops can be met by common varieties which are widely grown and produced in volume. However, there exists at present a growing market for crops with special end-use properties which are not met by grain of standard composition. Most commonly, specialty maize is differentiated from xe2x80x9cnormalxe2x80x9d maize, also known as field corn, by altered endosperm properties, such as an overall change in the degree of starch branching, as in waxy or high amylose maize, an increased accumulation of sugars as in sweet corn, or an alteration in the degree of endosperm hardness as in food grade maize or popcorn; Glover, D. V. and E. T. Mertz, 1987, Corn. In: Nutritional Quality of Cereal Grains; Genetic and Agronomic Improvement, R. A. Olson and K. J. Frey, eds. American Society of Agronomy, Madison, Wis., pp. 183-336; Rooney, L. W. and S. O. Serna-Saldivar, 1987, Food Uses of Whole Corn and Dry-Milled Fractions, In: Corn:Chemistry and Technology, S. A. Watson and P. E. Ramstead, eds. American Association of Cereal Chemists, Inc., St. Paul, Minn., pp. 399-429. xe2x80x9cSpecialtyxe2x80x9d crops are typically grown under contract for specific end users who place value on starch quality or other specific quality attributes. A specialty crop such as waxy maize is more valuable as a raw material to the starch industry than is normal or commodity grade maize, and thus is referred to as a value added crop. Currently the market size and added value of waxy maize is such that approximately 150,000 acres are grown in the United States. Farmers are paid a premium for growing specialty crops such as waxy maize because it is more valuable than normal maize and must not be mixed with normal maize. Because of the desire of many humans to eat a healthier diet and the documented effects of oleic acid on reducing cholesterol, the present invention will have greater value than normal corn. The current invention offers farmers the opportunity to grow a higher value crop than normal maize.
Oil is obtained from plants by a milling process. Corn oil is extracted from kernels through the use of a either a wet or dry milling process. Wet milling is a multi-step process involving steeping and grinding of the kernels and separation of the starch, protein, oil, and fiber fractions. A review of the maize wet milling process is given by S. R. Eckhoff in the Proceedings of the Fourth Corn Utilization Conference, Jun. 24-26, 1992, St. Louis, Mo., printed by the National Corn Growers Association, CIBA-GEIGY Seed Division and the United States Department of Agriculture. Dry milling is a process by which the germ and hull of the corn kernel are separated from the endosperm by the controlled addition of water to the grain and subsequent passage through a degerming mill and a series of rollers and sieves. The U.S. dry milling industry produces approximately 50 million pounds of crude corn oil per year, and the wet milling industry produces over one billion pounds of crude corn oil; Fitch, 1985, supra. The present invention offers the wet and dry milling industries the opportunity to process and sell a higher value oil than normal corn oil.
Applicants have discovered a corn plant capable of producing grain having a ten fold increase in oleic acid content over normal corn by breeding a high oil corn variety with a corn variety that carries a chemically mutated gene that confers high oleic acid content. Specifically, a corn plant has been bred to produce grain having 17 to 20% oil, with about 60% of the oil being oleic acid. Plants of this type can be used to pollinate high yielding, commercially acceptable hybrids that are male sterile, which have high oleic acid producing characteristics, thus producing grain having a five fold increase in oleic acid content over normal corn. By using this method and pollinator plants of this type, the timeline for commercial production of corn having elevated oleic levels is greatly accelerated.
This invention consists of corn grain produced by planting in close proximity, preferably in a field, corn plants of an agronomically elite high-yielding female parent, having high oleic characteristics, and optionally having high-oil characteristics, with corn plants of a high-oil and high oleic male parent, optionally having high-yielding characteristics and/or agronomically elite characteristics. The grain may then be all harvested or harvested selectively, for example, so that kernels produced by the female plants are harvested as grain. In the planting described, the preferred high-oil, high oleic male parent plant, when self or sib pollinated, is capable of producing kernels having a total oil content ranging from 7.5% to 20% of the total seed weight, measured at zero percent moisture and an oleic acid content of not less than about 55% of the total oil content of the seed. The agronomically elite female parent, when self or sib pollinated, is capable of producing kernels having a total oil content of between about 2 percent to about 7.5 percent, preferably at least about 6%, of the total seed weight, measured at zero percent moisture, wherein the oleic acid content is not less than about 55% of the total oil content.
The grain produced has as oleic acid content of about 4% to about 7% of the total seed weight.
The present invention further comprises a corn oil, produced from the grain described above, which has 50% to 120% greater oxidative stability than that of xe2x80x9cnormalxe2x80x9d corn oil where the oxidative stability is obtained without the addition of antioxidants. Such oil is useful in human and animal food, in cooking, and in industrial applications.
Also taught herein is a method of developing corn varieties with altered levels of fatty acid compositions, including oleic acid, comprising treatment of seeds or pollen with a chemical mutagen to produce mutant plants. Mutagens useful herein are selected from ethylmethanesulfonate and nitrosomethylurea. Also taught is a corn variety produced by such method having total oleic acid content of not less than about 55% of the total oil content of the seed, when measured at about zero percent moisture.
A further embodiment of the invention relates to the use of high oil, high oleic grain as an animal feed to improve meat quality, particularly in swine and poultry.
The present invention further comprises mutant corn lines B73OL and AEC272OL lines which bear the ATCC accession numbers 97026, 97027, from which the high oleic characteristics of the male and female plants that are crossed, as described above, are generated. The invention also describes the high oil, high oleic corn variety resulting from the cross, ASKC28xc3x97B73OL, which bears the ATCC accession number 97042.
Cultures of the following biological materials have been deposited with the following international depository(ies):
American Type Culture Collection (ATCC)
10801 University Boulevard, Manassas, Va. 20110-2209, U.S.A.
under conditions that satisfy the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.